Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia

Refined cancer models are required if researchers are to assess the burgeoning number of potential targets for cancer therapeutics in a clinically relevant context that allows a fast turnaround. Here we use tumor-associated genetic pathways to transform primary human epithelial cells from the epidermis, oropharynx, esophagus and cervix into genetically defined tumors in a human three-dimensional (3D) tissue environment that incorporates cell-populated stroma and intact basement membrane. These engineered organotypic tissues recapitulated natural features of tumor progression, including epithelial invasion through basement membrane, a complex process that is necessary for biological malignancy in 90% of human cancers. Invasion was rapid and was potentiated by stromal cells. Oncogenic signals in 3D tissue, but not 2D culture, resembled gene expression profiles from spontaneous human cancers. We screened 3D organotypic neoplasia with well-characterized signaling pathway inhibitors to distill a clinically faithful cancer gene signature. Multitissue 3D human tissue cancer models may provide an efficient and relevant complement to current approaches to characterizing cancer progression.     

The stacking of chloroplast thylakoids: Quantitative analysis of the balance of forces between thylakoid membranes of chloroplasts,and the role of divalent cations

An analysis is made of the van der Waals dispersion attractive forces and electrostatic repulsive forces between the grana thylakoid membranes of chloroplasts. These forces are determined for negatively charged surfaces with a pK_a value of 4.7 for a bulk pH of 7.0 with a range of mono- and divalent cation concentrations and intermembrane spacing in the range 10 to 80 Å. For equilibrium under dark conditions, it is concluded that either there is extensive electrostatic binding of divalent cations (Mg²⁺) to the negatively charged membrane groups (phospholipid, sulfolipid, and protein carboxyl), or a redistribution of these groups between stacked and unstacked regions must be invoked.     

Remodeling of the human papillomavirus type 11 replication origin into discrete nucleoprotein particles and looped structures by the E2 protein

The human papillomavirus (HPV) DNA replication origin (ori) shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundreds of base pairs. The HPV type 11 ori spans 103 bp and contains three palindromic E2 binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 ori binding protein. These sites are separated by 64 and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 DNA ori bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound, indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies on the dependence of looping on salt, ATP, and DTT using full-length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N-terminal domain and factors that may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed.     

谈药剂治螟

综合防治,是大家公认的正确治螟措施。关于药剂治螟在综合防治中的位置问题,过去曾引起各方面热烈讨论,作者谨就十余年来从实际工作中所得的一点体会,提出个人的偏见,借供讨论参考,尚希读者不吝指教。     

A modified fluorescent intercalator displacement assay for RNA ligand discovery

Fluorescent intercalator displacement (FID) is a convenient and practical tool for identifying new nucleic acid-binding ligands. The success of FID is based on the fact that it can be fashioned into a versatile screening assay for assessing the relative binding affinities of compounds to nucleic acids. FID is a tagless approach; the target RNAs and the ligands or small molecules under investigation do not need to be modified in order to be examined. In this study, a modified FID assay for screening RNA-binding ligands was established using 3-methyl-2-((1-(3-(trimethylammonio)propyl)-4-quinolinylidene)methyl)benzothiazolium (TO-PRO) as the fluorescent indicator. Electrospray ionization mass spectrometry (ESI-MS) results provide direct evidence that correlates the reduction in fluorescence intensity observed in the FID assay with displacement of the dye molecule from RNA. The assay was successfully applied to screen a variety of RNA-binding ligands with a set of small hairpin RNAs. Ligands that bind with moderate affinity to the chosen RNA constructs (A-site, TAR [transactivation response element], h31 [helix 31], and H69 [helix 69] were identified.     

Molecular Species Identification of Spiny Lobster Phyllosoma Larvae of the Genus Panulirus from the Northwestern Pacific

To identify lobster phyllosoma larvae of the genus Panulirus occurring in waters adjacent to Japan, genetic variation within and between 10 Indo-Pacific lobster species was investigated using restriction fragment length polymorphism (RFLP) analysis for the 1300-base pair mitochondrial cytochrome oxidase I (COI) gene. RFLP analysis using two endonucleases (AluI and TaqI) enabled discrimination of all species, including the P. longipes complex. The diagnostic DNA markers, supplemented with nucleotide sequence analysis, were applied to 44 mid- to late-stage phyllosoma larvae (7.4 to 27.7 mm in body length) collected in the northwestern Pacific. These samples were unexpectedly variable in species composition, comprising P. japonicus (n = 16), P. longipes bispinosus (21), P. longipes longipes (1), P. “aka” (1), and P. penicillatus (5). Comparison of larval size at similar stages revealed that P. l. bispinosus larvae were significantly larger than P. japonicus.     

Conditionally activated E7 proteins of high-risk and low-risk human papillomaviruses induce S phase in postmitotic, differentiated human keratinocytes

The productive program of human papillomaviruses (HPVs) in epithelia is tightly linked to squamous differentiation. The E7 proteins of high-risk HPV genotypes efficiently inactivate the pRB family of proteins that control the cell cycle, triggering S phase in suprabasal keratinocytes. This ability has until now not been demonstrated for the low-risk HPV-6 or HPV-11 E7 proteins. An inducible system in which HPV-16 E7 is fused to the ligand binding domain of the human estrogen receptor (ER) was described by Smith-McCune et al. (K. Smith-McCune, D. Kalman, C. Robbins, S. Shivakumar, L. Yuschenkoff, and J. M. Bishop, Proc. Natl. Acad. Sci. USA 96:6999-7004, 1999). In the absence of hormone, E7ER is cytoplasmic, and upon addition of 17beta-estradiol, it translocates to the nucleus. Using organotypic epithelial raft cultures developed from primary human keratinocytes, we show that 16E7ER promotes either S-phase reentry or p21cip1 accumulation in differentiated keratinocytes in a stochastic manner as early as 6 h postinduction with 17beta-estradiol. A vector expressing the ER moiety alone had no effect. These observations prove unequivocally that the E7 protein drives S-phase reentry in postmitotic, differentiated keratinocytes rather than preventing S-phase exit while the cells ascend through the epithelium. HPV-11 E7ER and, much less efficiently, HPV-6 E7ER also promoted S-phase reentry by differentiated cells upon exposure to 17beta-estradiol. S-phase induction required the consensus pRB binding motif. We propose that the elevated nuclear levels of the low-risk HPV E7 protein afforded by the inducible system account for the positive results. These observations are entirely consistent with the fact that low-risk HPV genotypes replicate in the differentiated strata in patient specimens, as do the high-risk HPVs.     

Downregulation of p57kip2 promotes cell invasion via LIMK/cofilin pathway in human nasopharyngeal carcinoma cells

The members of Rho family are well known for their regulation of actin cytoskeleton to control cell migration. The Cip/kip members of cyclin‐dependent (CDK) inhibitors have shown to implicate in cell migration and cytoskeletal dynamics. p57^kip2, a CDK inhibitor, is frequently down‐regulated in several malignancy tumors. However, its biological roles in human nasopharyngeal carcinoma (NPC) cells remained to be investigated. Here, we found p57^kip2 has nuclear and cytoplasm distributions and depletion of endogenous p57^kip2 did not change the cell‐cycle progression. Inhibition of cell proliferation by mitomycin C promoted FBS‐mediated cell migration and accompanied with the downregulation of ΔNp63α and p57^kip2, but did not change the level of p27^kip1, another CDK inhibitor. By using siRNA transfection and cell migration/invasion assays, we found that knockdown of p57^kip2, but not ΔNp63α, involved in promotion of NPC cell migration and invasion via decrease of phospho‐cofilin (p‐cofilin). Treatment with Y‐27632, a specific ROCK inhibitor, we found that dysregulation of ROCK/cofilin pathway decreased p‐cofilin expression and induced cell migration. This change of p‐cofilin induced actin remodeling and pronounced increase of membrane protrusions. Further, silence of p57^kip2 not only decreased the interaction between p57^kip2 and LIMK‐1 assayed by immunoprecipitation but also reduced the level of phospho‐LIMK1/2. Therefore, this study indicated that dysregulation of p57^kip2 promoted cell migration and invasion through modulation of LIMK/cofilin signaling and suggested this induction of inappropriate cell motility might contribute to promoting tumor cell for metastasis. J. Cell. Biochem. 112: 3459–3468, 2011. © 2011 Wiley Periodicals, Inc.     

Acid-Soluble Nucleotides in an Asporogenous Mutant of Bacillus subtilis

An asporogenous mutant of Bacillus subtilis Sp(-)H12-3, which is considered to have a block at stage 0, showed general growth characteristics similar to those of sporulating cultures. However, a sudden increase in the total amount of acid-soluble nucleotides observed at t(2) in sporulating bacteria was completely absent in this mutant. In sporulating cells, a marked increase in two nucleotides which were identified to be uridine diphosphate (UDP)-galactose and UDP-N-acetylglucosamine was noted, whereas UDP-glucose appeared to be accumulated in the mutant cells at t(2). No unusual nucleotides were found in the strains of B. subtilis examined. The possible role of these UDP derivatives in early stages of sporulation in B. subtilis is discussed.